Phosphate limited inducible promoter and a Bacillus expression system

ABSTRACT

An evolvable production strain of  B. subtilis  exhibiting continuous or high level expression during protein evolution is described. An evolved  Bacillus subtilis  pstS promoter facilitates screening and production of secreted proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/483,842, filedJun. 29, 2004, which is the National Stage of International ApplicationNo. PCT/US02/23829, filed Jul. 26, 2002, which claims the benefit ofU.S. Provisional Application No. 60/308,922, filed Jul. 30, 2001.

FIELD OF THE INVENTION

The present invention provides methods and compositions of improvedBacillus expression systems. In particularly preferred embodiments, themethods and compositions further comprise a phosphate-limited induciblepromoter.

BACKGROUND OF THE INVENTION

One desired property for a Bacillus expression cassette is a strongpromoter, induced in stationary phase from a single gene copy. However,it was originally believed that a single gene expression system wouldnot deliver enough messages to saturate the expression machinery of theBacillus host. Thus, the current Bacillus production protocols have beendesigned such that amplification is utilized in order to create tandemgene repeats. Two problems typically arise from the use of theserepeats. First, genetic manipulation of tandem genes is very difficult.Consequently, protein engineering is performed in lab strains as singlecopy, then later moved from a lab strain into a production strain andamplified before testing. This causes delays in product development andis plagued by numerous concerns, including the differences between thecharacteristics of screen and production strains. Second, theamplification process used to make the repeats requires an antibioticmarker, which is not allowed for use in some production strains (e.g.,depending upon the product produced by the strains). Thus, there is aneed for improved Bacillus expression systems.

SUMMARY OF THE INVENTION

The present invention provides improved methods and compositions forBacillus expression systems. In preferred embodiments, the presentinvention provides evolvable production strains of B. subtilisexhibiting continuous or high level expression during protein evolution.In particularly preferred embodiments, the evolved B. subtilis pstSpromoter of the present invention facilitates screening and productionof secreted proteins.

In some particularly preferred embodiments, the evolved promoter of thepresent invention provides better specific productivity in low phosphatemedium than other stationary phase promoters (e.g., aprE), drives longterm production of relatively large amounts of protein duringfermentation, is not sensitive to comK, finds use as a single gene withno antibiotic marker, and finds use in production as a single oramplified gene. Furthermore, in some embodiments, the use of asporulation minus strain (e.g., spoIIe) prevents cells from entering thenon-productive spore state.

In some preferred embodiments, the present invention provides isolatednucleic acid comprising a B. subtilis PstS promoter variant. In someparticularly preferred embodiments, the present invention provides anisolated nucleic acid that encodes OS-6. In some alternativeembodiments, the present invention provides at least one B. subtilishost cell comprising nucleic acid comprising a B. subtilis PstS promotervariant. In still further embodiments, the present invention provideshost cells in which the B. subtilis PstS promoter variant nucleic acidis integrated into the chromosome of the host cell. In yet additionalembodiments, the present invention provides host cells that furthercomprise a nucleic acid encoding a polypeptide of interest under thetranscriptional control of the PstS promoter variant.

The present invention also provides expression constructs comprising anisolated nucleic acid encoding B. subtilis PstS promoter and a nucleicacid molecule encoding a polypeptide of interest.

The present invention further provides methods for controlling theexpression kinetics for a protein of interest, wherein preferred methodscomprise culturing the cells under phosphate limiting conditions. Inother embodiments, the present invention provides methods for producinga protein. In some preferred embodiments, the methods of the presentinvention comprise providing a host cell transformed with an expressionvector comprising nucleic acid encoding at least one PstS promotervariant, cultivating the transformed host cell under conditions suitablefor said host cell to produce the protein; and recovering the protein.

The present invention also provides methods for screening mutants cellsfor protein secretion (i.e., secretion of a protein of interest)comprising: providing a host cell transformed with an expression vectorcomprising PstS promoter; cultivating the transformed host cell underconditions suitable for the host cell to produce the protein in thepresence of a hydrolysable substrate; and measuring the extent ofhydrolysis of the substrate.

The present invention further provides Bacillus host cells capable ofexpressing a heterologous sequence under nutrient limited conditions. Insome preferred embodiments, the expression by Bacillus is prolonged.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction of the OS6 PstS promoter expressiondomain. The PstS promoter region (˜400 bp) was amplified by PCR from B.subtilis W168 chromosomal DNA using chimeric primers with endscomplementary to yhf and subtilisin flanking regions (OSPS-1 [SEQ IDNO:1] and OSPS-4 [SEQ ID NO:2]), using methods known in the art. Inaddition, two chromosome PCR products were generated using OS2chromosomal DNA as template, utilizing methods known in the art andprimers OSFN-5 (SEQ ID NO:3) and OSFN-4 (SEQ ID NO:4), as well as OSPS-5(SEQ ID NO:5) and OSBS-1 (SEQ ID NO:6). Three piece PCR fusions wereassembled and transformed into OS-1.1 (apr-Δ-OS1), using methods knownin the art, to produce the strain OS6. The result was a replacement ofthe aprE promoter of OS2 with 206 bp of the pstS promoter, correspondingto −100 to +106.

OSPS-1 (SEQ ID NO: 1) GTCTTTGCTTGGCGAATGTTCATCCATGATGTGGGCGTT OSPS-4(SEQ ID NO: 2) GACTTACTTAAAAGACTATTCTGTCATGCAGCTGCAATC OSFN-5(SEQ ID NO: 3) GGCAACCCCGACAGGCGTAAT OSFN-4 (SEQ ID NO: 4)GATGAACATTCGCCAAGCAAAGAC OSPS-5 (SEQ ID NO: 5) ACAGAATAGTCTTTTAAGTAAGTCOSBS-1 (SEQ ID NO: 6) ATATGTGGTGCCGAAACGCTCTGGGGTAAC

FIG. 2 shows productivity at higher densities for the new constructs. InPanel A, results are shown for cells grown overnight with varyingphosphate concentrations. In Panels B and C, results are shown for a 100μM phosphate MOPS 1 M overnight culture of OS6.31 used to inoculate MOPS1 M in 10 (open circle), 100 (open triangle), 300 (filled square) and500 (filled triangle) μM phosphate at 500 CFU/ml. Glucose, opticaldensity and protease productivity were monitored at the indicated times.

FIG. 3 provides an example of culture kinetics. A strain expressing ansecreted enzyme via the pstS promoter was inoculated into a culturecontaining a fluorescent substrate. As the strain grew, it consumedphosphate, eventually depleting it and therefore halting further growth.The phosphate limitation induced expression of the enzyme, resulting ina linear build up of enzyme in the culture. In response to enzymebuildup, substrate was cleaved to form product.

FIG. 4A provides data showing the productivity differences between aparent strain and a mutated hyperproducer. Shake-flasks (250 ml), filledwith 50 ml, 100 μMP Mops 1M were each inoculated with 5000 CFU/ml ofOS6.31 (squares) or EL13.2 (circles). OD (circles) and subtilisinconcentration (squares) were determined at the indicated time points.

FIG. 4B provides data depicting the increase in protease productionunder phosphate limited conditions.

FIG. 5A provides data showing substrate hydrolysis as a function ofbatched phosphate concentration in medium. In these experiments, four1536-well plates containing nutrient limited medium, suc-AF-AMCsubstrate and the indicated phosphate concentration (2 to 20 μM) wereinoculated with OS6 cells. AMC fluorescent plate averages weredetermined for each plate at 48 h incubation 37° C. (Y-axis).

FIG. 5B is a histogram indicating the frequency of protease producersunder various initial phosphate concentrations. As indicated, as theinitial phosphate concentration increased, the final cell mass was foundto proportionately increase.

FIG. 5C depicts the relationship between initial phosphate concentrationand protease concentration (filled circles) and substrate productreleased (open squares). In these experiments, the substrate, sAF-AMC,was cleaved by the protease, to release the fluorescent product, AMC.

FIG. 6A provides a schematic of the screening system used during thedevelopment of the present invention.

FIG. 6B provides a graph illustrating the evolution of thepstS/subtilisin expression cassette. Parental chromosome was amplifiedusing primers OSBS-1 (ATATGTGGTGCCGAAACGCTCTGGGGTAAC; SEQ ID NO:7) andOSBS-8 (CTTTTCTTCATGCGCCGTCAGCTTTTTCTC; SEQ ID NO:8) and Z-Taq™polymerase, resulting in a mutagenized PCR product (0.2% spontaneousmutation rate). The resulting PCR products were transformed intohypercompetent B. subtilis for integration into the Bacillus chromosomeby double crossover. Clones were plated into 1536-well plates containingphosphate limited medium plus a dipeptide substrate (sucAF-AMC).Following incubation, substrate hydrolysis was measured by AMCfluorescence. The light bars indicate the average AMC hydrolysis ofsubtilisin positive wells for the indicated screening round(approximately 10⁴ clones). Top producers were pooled and subjected tofurther rounds of Z-Taq™ mutagenesis; the screening results of thesesuccessive rounds are indicated on the graph. After round 4 of directedevolution, a single winner was selected and its behavior in 14-litermeasured (dark bars) was observed.

FIG. 7 provides a schematic of the various mutations (indicated by thelines within the construct) that were introduced into the promoter,signal sequence, propeptide or protein of interest (e.g., a protease).The mutations arose during sequential rounds of error prone PCR. Themutations depicted were cumulative and not the result of a single roundof mutagenesis.

FIG. 8 demonstrates that the hyper-producing strain depicted has alteredPstS expression.

FIG. 9 shows amplified pstS 14-liter production from a single or anamplified pstS promoter.

FIG. 10 is a graph comparing the protein production of a single copyevolved pstS promoter versus an amplified aprE promoter.

DESCRIPTION OF THE INVENTION

The present invention provides improved methods and compositions forBacillus expression systems. In particularly preferred embodiments, thepresent invention provides evolvable production strains of B. subtilisexhibiting continuous and/or high level protein expression duringprotein evolution. The evolved B. subtilis pstS promoter of the presentinvention facilitates screening and production of secreted proteins.

In some embodiments, the present invention provides methods forgenerating and screening populations of mutant microorganisms, inparticular Bacillus, the use of the pstS promoter to drive expression ofa heterologous protein, and a novel production microorganism. In apreferred embodiment the microorganism is a Bacillus species.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs (See e.g., Singleton,et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., JohnWiley and Sons, New York [1994], and Hale & Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, N.Y. [1991]). Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. The headings provided herein are notlimitations of the various aspects or embodiments of the invention.Accordingly, the terms defined immediately below are more fully definedby reference to the specification as a whole.

As used herein, the term “host cell” refers to a cell that has thecapacity to act as a host and expression vehicle for an incomingsequence (i.e., a sequence introduced into the cell), as describedherein. In one embodiment, the host cell is a microorganism. In apreferred embodiment, the host cells are Bacillus species.

As used herein, “Bacillus” refers to all species, subspecies, strainsand other taxonomic groups within the genus Bacillus, including, but notlimited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alcalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus, and B. thuringiensis.

As used herein, the term “DNA construct” refers to DNA that is used tointroduce nucleic acid sequences into a host cell or organism. The DNAmay be generated in vitro (e.g., by PCR) or any other suitabletechniques. In some preferred embodiments, the DNA construct comprises asequence of interest. The sequence of interest's nucleic acid isoperably linked to a promoter. In preferred embodiments, the promoter isthe pstS promoter. In some embodiments, the DNA construct furthercomprises at least one selectable marker. In further embodiments, theDNA construct comprises sequences homologous to the host cellchromosome. In other embodiments, the DNA construct includesnon-homologous sequences (See e.g., FIG. 1).

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene (i.e. the nucleic acid sequence which encodes agene product). In some embodiments, the coding region is present in acDNA form, while in other embodiments, it is present in genomic DNA orRNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. In someembodiments, suitable control elements (e.g., enhancers, promoters,splice junctions, polyadenylation signals, etc.) are placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, in some embodiments, the coding regionutilized in the expression vectors of the present invention containendogenous enhancers, splice junctions, intervening sequences,polyadenylation signals, or a combination of both endogenous andexogenous control elements.

As used herein, the terms “promoter,” “promoter element,” and “promotersequence,” refer to a DNA sequence which is capable of controlling thetranscription of the oligonucleotide sequence into mRNA when thepromoter is placed at the 5′ end of (i.e., precedes) an oligonucleotidesequence. Thus, a promoter is typically located 5′ (i.e., upstream) ofan oligonucleotide sequence whose transcription into mRNA it controls,and provides a site for specific binding by RNA polymerase and forinitiation of transcription.

The term “promoter activity” when made in reference to a nucleic acidsequence refers to the ability of the nucleic acid sequence to initiatetranscription of an oligonucleotide sequence into mRNA.

As used herein, the terms “nucleic acid molecule encoding,” “nucleotideencoding,” “DNA sequence encoding,” and “DNA encoding” refer to theorder or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA sequence thus codes for the amino acid sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isseparated from at least one contaminant nucleic acid with which it isordinarily associated in its natural source. Isolated nucleic acid isnucleic acid present in a form or setting that is different from that inwhich it is found in nature. In contrast, non-isolated nucleic acids arenucleic acids such as DNA and RNA which are found in the state theyexist in nature. For example, a given DNA sequence (e.g., a gene) isfound on the host cell chromosome in proximity to neighboring genes; RNAsequences, such as a specific mRNA sequence encoding a specific protein,are found in the cell as a mixture with numerous other mRNAs whichencode a multitude of proteins. However, isolated nucleic acid encodinga polypeptide of interest includes, by way of example, such nucleic acidin cells ordinarily expressing the polypeptide of interest where thenucleic acid is in a chromosomal or extrachromosomal location differentfrom that of natural cells, or is otherwise flanked by different nucleicacid sequence(s) than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. Isolated nucleic acid can be readily identified (if desired) by avariety of techniques (e.g., hybridization, dot blotting, etc.). At aminimum, when an isolated nucleic acid or oligonucleotide is to beutilized to express a protein the oligonucleotide contains the sense orcoding strand (i.e., the oligonucleotide may be single-stranded).Alternatively, it may contain both the sense and anti-sense strands(i.e., the oligonucleotide may be double-stranded).

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule.

As used herein, the term “structural gene” or “structural nucleotidesequence” refers to a DNA sequence coding for RNA or a protein whichdoes not control the expression of other genes. In contrast, a“regulatory gene” or “regulatory sequence” is a structural gene whichencodes products (e.g., transcription factors) which control theexpression of other genes.

As used herein, the term “wild-type” refers to a gene or gene productwhich has the characteristics of that gene or gene product when isolatedfrom a naturally occurring source. Typically, a wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Asused herein, the terms “wild-type sequence,” and “wild-type gene” areused interchangeably and refer to a sequence that is native or naturallyoccurring in a host cell. The wild-type sequence may encode either ahomologous or heterologous protein. A homologous protein is one the hostcell would produce without intervention. A heterologous protein is onethat the host cell would not produce but for the intervention.

In contrast, the term “modified” or “mutant” refers to a gene or geneproduct which displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

As used herein, the terms “mutant sequence,” and “mutant gene” are usedinterchangeably and refer to a sequence that has an alteration in atleast one codon occurring in a host cell's wild-type sequence. Inpreferred embodiments, the expression product of the mutant sequence isa protein with an altered amino acid sequence relative to the wild-type.In some embodiments, the expression product has an altered functionalcapacity (e.g., enhanced enzymatic activity).

As used herein, the terms “modified sequence” and “modified genes” areused interchangeably and refer to a deletion, insertion or interruptionof naturally occurring nucleic acid sequence. In some embodiments, theexpression product of the modified sequence is a truncated protein(e.g., if the modification is a deletion or interruption of thesequence). In some preferred embodiments, the truncated protein retainsbiological activity. In alternative embodiments, the expression productof the modified sequence is an elongated protein (e.g., if themodification is an insertion into the nucleic acid sequence). Inalternative embodiments, an insertion results in the production of atruncated protein as the expression product (e.g., if the insertionresults in the formation of a stop codon). Thus, it is contemplated thatinsertions result in either a truncated protein or an elongated proteinas an expression product, depending upon the character of the insertion.

In some preferred embodiments, mutant DNA sequences are generated withsite saturation mutagenesis in at least one codon. In other preferredembodiments, site saturation mutagenesis is performed for two or morecodons. In yet further embodiments, mutant DNA sequences have more than40%, more than 45%, more than 50%, more than 55%, more than 60%, morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 95%, or more than 98% homology with thewild-type sequence. Alternatively, in some embodiments, mutant DNA isgenerated in vivo using any known mutagenic procedure (e.g., radiation).The desired DNA sequence is then isolated and used in the methodsprovided herein. In some embodiments, the DNA constructs are wild-type,while in other embodiments, the constructs comprise mutant or modifiedsequences. These sequences may be homologous or heterologous. The terms“transforming sequence” and “DNA construct” are used interchangeablyherein.

As used herein, an “incoming sequence” means a DNA sequence that isnewly introduced into the host cell chromosome or genome. The sequencemay encode one or more proteins of interest. The incoming sequence maycomprise a promoter operably linked to a sequence of interest. In someembodiments, incoming sequences comprise sequence that is alreadypresent in the genome of the cell to be transformed, while in otherembodiments, it is not already present in the genome of the cell to betransformed (i.e., in some embodiments, it is homologous, while in otherembodiments, it is heterologous sequence).

In some embodiments, the incoming sequence encodes at least oneheterologous protein, including, but not limited to hormones, enzymes,growth factors. In some preferred embodiments, the incoming sequenceencodes at least one enzyme including, but not limited to hydrolases,proteases, esterases, lipases, phenol oxidases, permeases, amylases,pullulanases, cellulases, glucose isomerases, laccases, and proteindisulfide isomerases.

In an alternative embodiment, the incoming sequence encodes a functionalwild-type gene or operon, a functional mutant gene or operon, or anon-functional gene or operon. In some embodiments, the non-functionalsequence is inserted into a target sequence to disrupt function, therebyallowing a determination of function of the disrupted gene.

As used herein, the term “flanking sequence,” refers to any sequencethat is either upstream or downstream of the sequence being discussed(e.g., for genes A B C, gene B is flanked by the A and C genesequences). In some preferred embodiments, the incoming sequence isflanked by a homology box on each side. In one more preferredembodiment, the incoming sequence and the homology boxes comprise a unitthat is flanked by stuffer sequence (as defined below) on each side.While a flanking sequence may be present on only a single side (either3′ or 5′), in preferred embodiments, flanking sequences are present oneach side of the sequence being flanked.

As used herein, “stuffer sequence” refers any extra DNA that flanks thehomology boxes. In most cases, these are typically vector sequences.However, these sequences are contemplated to be any non-homologous DNAsequence. Indeed, a stuffer sequence provides a non-critical target fora cell to initiate DNA uptake. It is not intended that the presentinvention be limited to any specific mechanism or sequence.

As used herein, the term “homology box” refers to the sequences flankinga sequence of interest. In preferred embodiments, the sequence of eachhomology box is homologous to a sequence in the Bacillus chromosome.These sequences direct where in the Bacillus chromosome the newconstruct gets integrated and what part of the Bacillus chromosome isreplaced by the incoming sequence.

As used herein, the term “homologous recombination” refers to theexchange of DNA fragments between two DNA molecules or pairedchromosomes (i.e., during crossing over) at the site of identicalnucleotide sequences. In a preferred embodiment, chromosomal integrationis accomplished via homologous recombination.

As used herein, the term “homologous sequence” refers to a sequence thatis found in the same genetic source or species as the host cell. Forexample, the host cell strain may be deficient in a specific gene. Ifthat gene is found in other strains of the same species the gene wouldbe considered a homologous sequence.

As used herein, the term “heterologous sequence” refers to a sequencederived from a different genetic source or species than the host cell.In some embodiments, a heterologous sequence is a non-host sequence,while in other embodiments, it is a modified sequence, a sequence from adifferent host cell strain, or a homologous sequence from a differentchromosomal location of the host cell.

The term “transfection” as used herein refers to the introduction offoreign DNA into cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, biolistics (i.e., particlebombardment) and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to “polynucleotides” and “oligonucleotides” (which areinterchangeable terms that refer to a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “5′-CAGT-3′,” iscomplementary to the sequence “5′-ACTG-3′.” Complementarity can be“partial” or “total.” “Partial” complementarity is where one or morenucleic acid bases is not matched according to the base pairing rules.“Total” or “complete” complementarity between nucleic acids is whereeach and every nucleic acid base is matched with another base under thebase pairing rules. The degree of complementarity between nucleic acidstrands may have significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This may be of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

As used herein, the term “chromosomal integration” refers to the processwhereby the incoming sequence is introduced into the chromosome (i.e.,genome) of a host cell. In some particularly preferred embodiments ofthe present invention, the incoming sequence is introduced into theBacillus chromosome. In this process, the homology boxes of thetransforming DNA align with homologous regions of the chromosome.Subsequently, the sequence between the homology boxes is replaced by theincoming sequence in a double crossover (i.e., homologousrecombination).

As used herein, the term “selectable marker” refers to the use of any“marker” (i.e., indicator), which indicates the presence or absence of aprotein or gene of interest. In some embodiments, the term encompassesgenes which encode an enzymatic activity that confers the ability togrow in medium lacking what would otherwise be an essential. In otherembodiments, a selectable marker confers resistance to an antibiotic ordrug upon the cell in which the selectable marker is expressed. Inaddition, selectable markers include markers (e.g., genes) that conferantibiotic resistance or a metabolic advantage to the host cell, suchthat cells containing exogenous DNA are distinguishable from cells thathave not received any exogenous sequence during the transformation. A“residing selectable marker” is one that is located on the chromosome ofthe microorganism to be transformed.

“Amplification” is defined herein as the production of additional copiesof a nucleic acid sequence and is generally carried out using polymerasechain reaction and other technologies that are well known in the art. Asused herein, the term “polymerase chain reaction” (“PCR”) refers to themethods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, all ofwhich are hereby incorporated by reference, which describe a method forincreasing the concentration of a segment of a target sequence in a DNAsample (e.g., genomic DNA) without cloning or purification. The lengthof the amplified segment of the desired target sequence is determined bythe relative positions of two oligonucleotide primers with respect toeach other, and therefore, this length is a controllable parameter.Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;or incorporation of ³²P-labeled deoxynucleotide triphosphates, such asdCTP or dATP, into the amplified segment). In addition to genomic DNA,any oligonucleotide sequence can be amplified with the appropriate setof primer molecules. In particular, the amplified segments created bythe PCR process itself are, themselves, efficient templates forsubsequent PCR amplifications.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and of an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that it is detectable in any detection system, including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double- orsingle-stranded DNA at or near a specific nucleotide sequence.

The Pho Network

The Pho network in Bacillus consists of many components and affects allcell machinery. The Pho regulon of B. subtilis includes the structuralgenes for three secreted alkaline phosphatases (Apases): phoA—expressedprimarily during phosphate starvation; phoB expressed from tandempromoters either during phosphate starvation or during stage II of sporedevelopment; phoD expressed during phosphate starvation and encoding anenzyme with alkaline phosphodiesterase activity as well as APaseactivity. The phoD alkaline phosphatases also has a putative role incell wall teichoic acid turnover during phosphate deprivation.

Other Pho genes include: the tuaABCDEFGH operon, which is responsiblefor synthesis of an anionic cell wall polymer, teichuronic acid(P-free); tagAB, tag DEF divergon, responsible for synthesis of teichoicacids (poly(glycerol phosphate)) of cell walls; pstSACB1B2 genesencoding the phosphate transport system; and the phoPR operon. InBacillus genome databases, pst genes have been referred to usingdifferent names (pstS=yqgG, pstC=yqgH, pstA=yqgI, pstB1=yqgJ,pstB2=yqgK). Nonetheless, the Pho regulon offers several strong,regulated promoters, which can be used alone or in combination forphosphate regulated expression of genes of interest. The criticalfeature is the use of appropriate media and growth conditions to utilizethe full potential of these and other inducible Bacillus promoters.Other useful promoters are those induced under stressful environmentalconditions (i.e., the induction of specific starvation responses, suchas the stringent response and the general starvation response). Forexample, U.S. Pat. No. 6,175,060 describes the control of plantexpression patterns involving a phosphate-depleted inducible promoterand phosphate limitation.

U.S. Pat. No. 5,304,472 describes the control of the extent and rate ofproduction of E. coli expression by mutations in PstS such thatphosphate induced promoters induce under non-starvation conditions. Thispatent indicates that polypeptide transcription is driven by the phoApromoter and expression is controlled by varying the phosphateconcentration in the culture medium. This patent further indicates thatphosphate starvation interferes with protein expression in E. coli.Unlike, B. subtilis which has evolved to produce proteins underphosphate limiting conditions, the E. coli pstS system requires nonstationary production. Thus, it is contemplated that the processdescribed in this patent will not find use in B. subtilis productionsystems.

U.S. Pat. No. 5,789,199 suggests removing the native pstS gene from E.coli and using variant pstS gene products to facilitate heterologousprotein production. Over-expression of DsbA or DsbC is said tofacilitate disulfide arrangement. The mutations described in U.S. Pat.No. 5,789,199 and U.S. Pat. No. 5,304,472 lie in the pstS coding regionand are targeted to increase pstS expression under non-phosphatelimiting conditions

In most embodiments of the present invention, sporulation negativemutants are preferred to sporulation competent strains. Any sporulationnegative mutant may be used in the present invention. In someembodiments, a preferred sporulation negative mutant is spoIIe⁻.

Construct Assembly

In one general embodiment, the present invention involves assembling aDNA construct in vitro, followed by direct cloning of such constructinto competent Bacillus cells, such that the construct becomesintegrated into the Bacillus genome. For example, in some embodimentsPCR fusion and/or ligation are employed to assemble a DNA construct invitro. In a preferred embodiment, the DNA construct is a non-plasmid DNAconstruct. In another embodiment, the DNA construct comprises a DNA intowhich a mutation has been introduced. This construct is then used totransform Bacillus cells. In this regard, highly competent mutants ofBacillus are preferably employed to facilitate the direct cloning of theconstructs into the cells. For example, Bacillus carrying the comK geneunder the control of a xylose-inducible promoter (Pxyl-comK) can bereliably transformed with very high efficiency, as described herein. Anysuitable method known in the art may be used to transform the cells. TheDNA construct may be inserted into a vector (i.e., a plasmid), prior totransformation. In some preferred embodiments, the circular plasmid iscut using an appropriate restriction enzyme (i.e., one that does notdisrupt the DNA construct). Thus, in some embodiments, circular plasmidsfind use with the present invention. However, in alternativeembodiments, linear plasmids are used. In some embodiments, the DNAconstruct (i.e., the PCR product) is used without the presence ofplasmid DNA.

In some preferred embodiments, libraries of mutants are generated. It iscontemplated that the library of mutants be screened according to themethods provided herein. However, any appropriate method known to thoseskilled in the art will find use with the present invention. Generally,the inventive methods involve the isolation of various host cellcultures for the purpose of selecting higher expression and/or secretionof the protein of interest. Prior productivity screens were poorpredictors of which mutants would demonstrate enhanced proteinproduction in large scale fermentations. Thus, the present inventionfulfills a need in the art for improved screening and productionmethods.

In a general aspect, the screening methods described herein provide fastand predictive methods for identifying clones of interest. In someembodiments, a host cell library (i.e., host cells transformed with aDNA construct) is plated at a concentration of 0.5 CFU/well in 1536-wellplates with a slowly hydrolysable substrate (e.g., sAFAMC). The cellsare cultured at least 48 hours to assay for robust long-termproductivity. Contents from the five wells with the highest signal(e.g., fluorescence) level are assayed in a secondary screen (e.g.,using sAAPFpNA), to confirm primary screen activity. The secondaryscreen is essential in eliminating potential false-positive results byusing statistical analysis concerning large number distributions.

Cells that demonstrate enhanced productivity of the protein of interestusing the screening methods provided for herein also possess enhancedprotein production in large scale production methods, as well asprolonged protein secretion. The use of the nutrient limited induciblepromoters provides methods of controlling expression kinetics, which arecontemplated to assist in screening.

EXPERIMENTAL

The following examples are illustrative and are not intended to limitthe present invention.

In the experimental disclosure which follows, the followingabbreviations apply: C (degrees Centigrade); rpm (revolutions perminute); H₂O (water); aa (amino acid); by (base pair); kb (kilobasepair); kD (kilodaltons); gm, G, and g (grams); μg and ug (micrograms);mg (milligrams); ng (nanograms); μl and ul (microliters); ml(milliliters); mm (millimeters); nm (nanometers); μm and um(micrometer); M (molar); mM (millimolar); μM and uM (micromolar); U(units); V (volts); OD₆₂₀ (optical density at 620 nm); MW (molecularweight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours);MgCl₂ (magnesium chloride); NaCl (sodium chloride); PAGE (polyacrylamidegel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2]); PCR (polymerase chain reaction); PEG(polyethylene glycol); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); w/v (weight to volume); v/v (volumeto volume); Amersham (Amersham Pharmacia Biotech, Arlington Heights,Ill.); ICN (ICN Biomedicals, Inc., Aurora, Ohio); ATCC (American TypeCulture Collection, Rockville, Md.); BioRad (BioRad, Richmond, Calif.);Clontech (CLONTECH Laboratories, Palo Alto, Calif.); GIBCO BRL or GibcoBRL (Life Technologies, Inc., Gaithersburg, Md.); Invitrogen (InvitrogenCorp., San Diego, Calif.); Kodak (Eastman Kodak Co., New Haven, Conn.);New England Biolabs (New England Biolabs, Inc., Beverly, Mass.); Novagen(Novagen, Inc., Madison, Wis.); Pharmacia (Pharmacia, Inc., Piscataway,N.J.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Sorvall (SorvallInstruments, a subsidiary of DuPont Co., Biotechnology Systems,Wilmington, Del.); Stratagene (Stratagene Cloning Systems, La Jolla,Calif.); Qiagen (Qiagen, Valencia, Calif.); Perkin Elmer (Perkin Elmer,Wellesley, Mass.); and PE/ABI (Perkin Elmer/Applied Biosystems, FosterCity, Calif.).

Example 1 Construction of OS6 PstS Promoter Expression Domain andTransformation in Bacillus

A fragment containing the pstS promoter was obtained by PCR usingprimers OSPS-1 (SEQ ID NO:1) and OSPS-4 (SEQ ID NO:2) and chromosomalDNA from B. subtilis strain W168. A second fragment containing thesubtilisin gene was obtained by PCR using primers OSPS-5 (SEQ ID NO:3)and OSBS-1 (SEQ ID NO:6) and OS2 chromosomal DNA. A third fragmentcontaining upstream chromosomal sequences was obtained by PCR usingprimers OSFN-5 (SEQ ID NO:5) and OSFN-4 (SEQ ID NO:4) and OS2chromosomal DNA. These fragments provided the preferred overlaps of atleast 15 nucleotides for assembly. Indeed, in a majority of theexperiments used in the development of the present invention, 18-20nucleotide overlaps were used. After purification using standard methodsknown in the art, the fragments were fused together using methods wellknown in the art. The primer sequences used are shown below:

OSPS-1: (SEQ ID NO: 1) GTCTTTGCTTGGCGAATGTTCATCCATGATGTGGGCGTT OSPS-4:(SEQ ID NO: 2) GACTTACTTAAAAGACTATTCTGTCATGCAGCTGCAATC OSFN-5:(SEQ ID NO: 3) GGCAACCCCGACAGGCGTAAT OSFN-4: (SEQ ID NO: 4)GATGAACATTCGCCAAGCAAAGAC OSPS-5: (SEQ ID NO: 5) ACAGAATAGTCTTTTAAGTAAGTCOSBS-1: (SEQ ID NO: 6) ATATGTGGTGCCGAAACGCTCTGGGGTAAC

A typical PCR reaction (100 μl) contained 1×Pfu Buffer, 1.5 μl 10 mMdNTPs, 1 μl 25 μM primer, 1 μl Pfu Turbo® DNA polymerase, and 200 ngplasmid DNA. The cycling conditions were: 95° C. for 35 seconds for onecycle, followed by 16 cycles of 95° C. for 35 seconds, 50° C. for 1minute, 68° C. for 16.5 minutes. Following cycling, the reactionmixtures were held at 68° C. for 7 minutes.

The final PCR product (i.e., the DNA construct), is shown in FIG. 1. ABacillus strain (OS-1.1 [apr—OS1]) was directly transformed with the PCRproduct. The transformed cells were designated “OS6.” The result was areplacement of the aprE promoter of OS2 with 206 bp of the pstSpromoter, corresponding to nucleotides −100 to +106.

Thus, the aprE promoter of subtilisin was replaced with the −100 to +106region of the pstS promoter from B. subtilis W168 (FIG. 1). As shown inTable 1 below, the resulting strain produces subtilisin when the freephosphate in the culture becomes growth limiting.

TABLE 1 Phosphate-Limited Subtlisin Expression in OS6 Turbidity TiterMedium (OD 620 nm) (mg/l subtilisin) Rich 3.3 0 Phosphate Limited 0.1113

Thus, in rich media, cell growth and expression are limited by oxygen orglucose, the pstS promoter remains repressed and no subtilisin isexpressed because phosphate is not depleted. In contrast, underconditions where the concentration of phosphate in the medium limitsgrowth, subtilisin expression is detected.

Example 2 Phosphate Limited Productivity

Growth under limited phosphate conditions provides uniform proteinproduction, as described herein. FIG. 2 provides graphs showing growthand subtilisin production for a 100 μM phosphate MOPS 1M (see followingpage for Table 2, which provides the formulae used) overnight culture ofOS6.31 used to inoculate MOPS 1 M in 10 (open circle), 100 (opentriangle), 300 (filled square) and 500 (filled triangle) μM phosphate at500 CFU/ml. Glucose, optical density and protease productivity weremonitored at the times indicated in FIG. 2.

As indicated in FIG. 2, Panel A, the phosphate concentration may be usedto adjust stationary cell density. However, care must be taken to avoidhigh cell densities, because limitations on the oxygen or glucoseconcentrations (See, FIG. 2, Panel B) and/or medium nutrients may affectproductivity. FIG. 2, Panel C depicts the loss of productivity observedat higher cell densities.

As indicated in FIG. 3, when properly adjusted, cell density andproductivity remain relatively constant and predictable. The resultsshown in FIG. 3 were obtained from a strain expressing an secretedenzyme via the pstS promoter, which was inoculated into a culturecontaining a fluorescent substrate. As this strain grows, it consumesphosphate, thereby depleting the phosphate concentration in the medium,and eventually halting further growth. The phosphate limitation inducesexpression of the enzyme, resulting in a linear build up of enzyme inthe culture. In response to enzyme buildup, the substrate is cleaved toform product.

As indicated in Table 3 below, as compared with two other stationaryphase promoters, the pstS promoter provides better expression inphosphate limiting conditions.

TABLE 2 MOPS Formulae: MOPS 1M Volume or weight Solution FinalConcentration 10X Metals{circumflex over ( )} 100 ml 10X MOPS 1X 25.8G/L Na Citrate 14.4 ml 50% Glucose 40 mM 43.5 G/L K2SO4 10 ml 1M(NH₂)SO₄ 10 mM — 0.038 ml 0.132M K2HPO4 5 uM 1.69 G/L MnSO4*H20 1 ml 1MCaCl2 1.1 mM* 13.2 G/L MgSO4*7H20 60.38 gm Na2SO4*10H2O 187.5 mM 10.89gm K2SO4 62.5 mM 100 mls 10X Metals^(#) 0.5 ml 10 mg/ml Chloramphenicol5 ug/ml H20 100 ul 100 mg/ml soytone 10 ppm (5 uM P, 40 mM Glucose, 250mM Na Sulfate, 1.1 mM CaCl2, Ammonia, and metals w/o FeSO4) 1 literfinal Volume The pH should be adjusted to 7.3 after all components areadded and dissolved Filter sterilize 0.22 micron filter *There is 0.1 mMCaCl2 in the 1X MOPS ^(#)see recipes beside Table {circumflex over( )}Based on MOPS 1L, lower Fe reduces the absorbance in the 405 nmrange, but with ammonia instead of urea

TABLE 3 Mutations that Improve Subtilisin Expression. 96-well¹fermentor² Strain ppm g/l Mutations³ OS6.31 2 0.6 parent strain EL13.24.4 2.1 A17T, A32G, T86C, G189A [V9I], G233A, A369G (I69V), T417C,T464G, A489G (T109A) EL13.3 4.6 n.d. A17T, A26G, A32G, G189A [V9I],G233A, A369G (I69V), T417C, A489G (T109A) ¹48 h in 10 μM phosphate MOPS1M medium ²Phosphate limited, glucose limited defined medium 14-literfermentation ³Mutations are relative to +1 pstS position GTAGGACAA (SEQID NO: 9); changes in subtilisin signal sequence are indicated inbrackets, residues changes in the mature subtilisin molecule areindicated in parentheses.

As shown in FIG. 10, under conditions where phosphate is not growthlimiting, the pstS promoter drives less productivity than aprE.

Example 3 Shake Flask Productivity

Predictable stationary productivity facilitates diversity screening.FIG. 4 provides one example of a screen for detecting mutants withimproved secretion and activity. A strain expressing a secreted enzymevia the pstS promoter was inoculated into a culture containing afluorescent substrate. As the strain grew, phosphate was consumed andeventually depleted, halting further growth. The phosphate limitationinduced expression of the enzyme, resulting in a linear build up ofenzyme in the culture. In response to enzyme buildup, the substrate wascleaved, resulting in the production of product.

Example 4 Controlled Enzyme Productivity

The dependence of the pstS promoter on phosphate limitation provides asimple method for controlling screening kinetics. In these experiments,cells were grown overnight in MOPS 1M and then diluted to 50 cells/mL or25,000 cells total, in 510 mL media. Cells were plated onto 1536-wellmicrotiter plates with MOPS 1M containing SAF-AMC substrate to provide afinal concentration of 7.5 μM. All plates were placed in a humidifiedincubator set at 37° C. and allowed to grow for 48 hours. The plateswere removed and read using a Perkin Elmer HTS 7000 Plus.

FIG. 5 demonstrates the ability to adjust the AMC hydrolysis in an endpoint assay by altering the starting phosphate concentration in themedium. The more batched phosphate, the greater the final cell density,and the more secreted product. Controlling the batched phosphate allowedfor a greater than 7-fold range of protein expression, which in turnprovides additional flexibility in screen development.

Example 5 Expression Cassette Mutagenesis

As described in this Example and shown in the Figures, mutagenesis ofthe pstS expression cassette is useful in production of mutations thatimprove expression. For example, FIG. 6 depicts the 2-fold increase inexpression observed through successive rounds of mutagenesis, whilestill maintaining phosphate regulation (Table 4).

TABLE 4 Evolved pstS Promoter Remains Phosphate Regulated Strain 10 μM 5mM Parent 3.0 0.6 EL13.2 9.4 1.0

These strains were grown overnight on LA+5 μM CMP plates, the colonieswere then transferred into 100 μM phosphate MOPS medium in a 96-wellplate, and incubated overnight at 37° C. with shaking, in a humidifiedbox. Then, a 1/121 dilution of this culture was used to inoculate MOPSmedium containing either 10 μM or 5 mM phosphate contained within96-well plates. After 55 hours of incubation at 37° C. with shaking,protease activity was measured by determining the hydrolysis ofSuc-AAPF-PNA by a culture sample.

To randomly mutagenize the signal sequence and propeptide of subtilisingene, a PCR reaction using primers OSFN-5 (SEQ ID NO:3) and OSFN-4 (SEQID NO:4) generated the 1.9 Kb left flanking region (i.e., the yhfQ-P-Oregion). Primers OSPS-1 (SEQ ID NO:1) and OSPS-4 (SEQ ID NO:2) were usedto mutate a 646 bp region comprising the promoter, signal sequence andpropeptide region. Primers OSPS-5 (SEQ ID NO:5) and OSBS-1 (SEQ ID NO:6)were used to generate the 5.1 kb right flanking region (i.e., theyhfN-M-L region). Primers OSFN-4 (SEQ ID NO:4) and OSPS-1 (SEQ ID NO:1)and primers OSPS-4 (SEQ ID NO:4) and OSPS-5 (SEQ ID NO:5) arecomplementary to one another. A typical amplification reaction (100 μl)was set up using either 0.5 μM of primers OSFN-5 (SEQ ID NO:3) andOSFN-4 (SEQ ID NO:4) for the 1.9 Kb fragment, or 0.5 μM of primersOSPS-5 (SEQ ID NO:5) and OSBS-1 (SEQ ID NO:6) for the 5.1 Kb fragment.To this mixture, 200 μM dNTP, 2 μl of log phase liquid culture grown toOD₆₀₀=0.5 (source of Bacillus chromosomal DNA), 4U rTth XL polymerase,1.25U Pfu Turbo® DNA polymerase, 1×rTth XL polymerase buffer and 1.1 mMMg (OAc)₂.

The amplification parameters for the 2.2 Kb and 3.9 Kb fragments were:95° C. for 3 min, 95° C. for 30 sec, 54° C. for 30 sec, and 68° C. for 2min for a total of 30 cycles.

The PCR reaction products were analyzed on an agarose gel. If thecorrect size fragment was observed, then the PCR product was purifiedusing the QIAquick™ PCR purification kit (Qiagen), per themanufacturer's instructions.

The 646 bp fragment for mutagenizing the maturation site was amplifiedusing Primers OSFN-4 (SEQ ID NO:4) and OSPS-4 (SEQ ID NO:2) (0.5 μMeach), 33 μl 3×dNTP, 2 μl of liquid culture grown to OD₆₀₀=0.5 (i.e.,the source of Bacillus chromosomal DNA), 0-0.3 mM MnCl₂ (varies upon therate of mutagenesis desired), 5.5 mM MgCl₂, 5U Taq polymerase, and 1×Taqpolymerase buffer in a 100 μl reaction. The PCR amplification parameterswere as follows: 95° C. for 30 sec, 54° C. for 30 sec, and 68° C. for 30sec for a total of 30 cycles. The PCR reaction products were analyzed onan agarose gel. If the correct size fragment was seen, the PCR productwas purified using the QIAquick™ PCR Purification Kit, using the kitmanufacturer's instructions.

The assembly of the entire 6.8 kb fragment containing the mutagenizedmaturation site was done using 3-5 ul each of 646 bp, 1.9 kb, and 5.1 kbfragments, 0.5 μM each of Primers OSFN-5 (SEQ ID NO:3) and OSBS-1 (SEQID NO:6), 300 μM dNTP, 4U r XL polymerase, 1.25U Pfu Turb® DNApolymerase, 1×rTth XL polymerase buffer, and 1.1 mM Mg (OAc)₂ in a 100ul reaction. The parameters for the assembly reaction were as follows:95° C. for 30 sec, 48-50° C. for 30 sec, and 68° C. for 7 min. for atotal of 30 cycles. The PCR reaction products were analyzed on anagarose gel. If the correct size fragment was seen, the PCR product wastransformed into Pxyl-comK Bacillus strains to generate a library.

Example 6 Predictive Screening Assay

Importantly, the evolved pstS promoter produces more product during14-liter fermentation (See, FIG. 6, filled bars), demonstrating that thescreen predicted production activity. Some of these mutations lie withinthe promoter and untranslated region of the expression cassette (See,FIG. 7). Some of the mutations lie within the pho box, a region in whichphoA binds. Transcript analysis of one of the most evolved mutants(EL13.2) indicates an altered transcription pattern (See, FIG. 8).

Parental chromosome was amplified using primers OSBS-1 (SEQ ID NO:1) andOSBS-8 (SEQ ID NO:8) and Z-Taq™ polymerase, resulting in a mutagenizedPCR product (0.2% spontaneous mutation rate). The resulting PCR productswere transformed into hypercompetent B. subtilis for integration intochromosome by double crossover. Clones were plated into 1536-well platescontaining phosphate limited medium plus a dipeptide substrate(sucAF-AMC). Following incubation, substrate hydrolysis was measured byAMC fluorescence. The light bars in FIG. 6B indicate the average AMChydrolysis of subtilisin positive wells for the indicated screeninground (approximately 10⁴ clones). Top producers were pooled andsubjected to further rounds of Z-Taq™ mutagenesis; the screening resultsof these successive rounds are indicated on the graph. After round 4 ofdirected evolution, a single winner was selected and its behavior in14-liter measured (dark bars).

Example 7 Promoter Amplification

As shown in FIG. 9, amplification of the evolved pstS promoter improvesexpression during fermentation. In this case, the amplification did notimprove the specific productivity, but production began 4 hours earlierthan with the single copy gene. An additional advantage of this promoteris that production continues longer than with amplified aprE drivenexpression. FIG. 10 depicts the production continuing past 60 hourswhereas the aprE promoter has ceased production by 40 hours. Thus, thepresent invention provides means for production of the protein ofinterest for a prolonged period.

Although the foregoing describes a phosphate limited inducible promoter,the above techniques are suitable for use with any nutrient limitedinducible promoter. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to any specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artand in fields related thereto are intended to be within the scope of thefollowing claims.

The invention claimed is:
 1. A method for controlling the expressionkinetics of a protein of interest, said method comprising culturingunder phosphate limiting conditions, a Bacillus host cell comprising anucleic acid comprising a Bacillus subtilis PstS promoter variant, saidPstS promoter variant comprising a sequence corresponding to nucleotides−100 to +106 of PstS promoter from B. subtilis strain W168, wherein saidsequence comprises at least one mutation chosen from A17T, A26G, A32Gand T86C.
 2. The method of claim 1, wherein said nucleic acid isintegrated into the chromosome of said host cell.
 3. The method of claim1, wherein said host cell further comprises a nucleic acid encoding apolypeptide of interest under the transcriptional control of said PstSpromoter variant.
 4. The method of claim 1, wherein said host cellexpresses a heterologous sequence under nutrient limited conditions. 5.The method of claim 4, wherein said expression is prolonged.
 6. A methodof producing a protein, comprising: providing a host cell transformedwith an expression vector comprising a nucleic acid comprising aBacillus subtilis PstS promoter variant, said PstS promoter variantcomprising a sequence corresponding to nucleotides −100 to +106 of PstSpromoter from B. subtilis strain W168, wherein said sequence comprisesat least one mutation chosen from A17T, A26G, A32G and T86C; cultivatingsaid transformed host cell under conditions suitable for said host cellto produce said protein; and recovering said protein.
 7. A method ofscreening mutants cells for protein secretion comprising: providing ahost cell transformed with an expression vector comprising a nucleicacid comprising a Bacillus subtilis PstS promoter variant, said PstSpromoter variant comprising a sequence corresponding to nucleotides −100to +106 of PstS promoter from B. subtilis strain W168, wherein saidsequence comprises at least one mutation chosen from A17T, A26G, A32Gand T86C; cultivating said transformed host cell under conditionssuitable for said host cell to produce said protein in the presence of ahydrolysable substrate; and measuring the extent of hydrolysis of thesubstrate.